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In this study, the stereoisomers of two tetracyclic quinolines previously discovered as Pregnane X Receptor ligands were stereodiscriminated with a Chiralcel OD‐H column under normal‐phase conditions. The two compounds are characterized by a C/D cis ring junction and the co‐existence of four diastereoisomers for the presence of a further chiral center at the ring C. A mobile phase made‐up of n‐hexane/2‐PrOH – 99:1 (v/v) allowed to obtain the four peaks of each stereoisomer fully resolved with enantioseparation and enantioresolution factor values up to 2.98 and 11.16, respectively. Due to the lack of pure enantiomeric standards of known stereochemistry, an indirect protocol was successfully applied to define the enantiomeric elution order (EEO) under the identified optimal eluent conditions. This was possible by comparing experimental and theoretical Time‐Dependent Density Functional Theory (TD‐DFT) electronic circular dichroism spectra. In order to confirm the EEOs established through the ab initio calculations, molecular docking simulations were successfully performed with a nonameric model structure of the chiral selector. The results from the docking simulations were in perfect accordance with the results from the TD‐DFT study, thereby confirming the importance of performing multiple methods in the case of indirect attribution of the absolute configuration. [ABSTRACT FROM AUTHOR]

The capacity of nonsteroidal antiinflammatory drugs (NSAIDs) to prevent prostanoids biosynthesis through the inhibition of COX-2 enzyme is related to their structural backbone, based on the fusion of a cis-stilbene unit with a variety of heterocyclic and carbocyclic rings. By this route, a series of new selective COX-2 inhibitors was developed, by maintaining the 4-methylsulfone or 4-methylsulfonamide substituent on the phenyl moiety, essential for their activity. In this frame, two novel propyl sulfoxide derivatives were synthesized, which proved selective and sufficiently potent COX-2 inhibition activity when tested as racemates. In the present study, the use of a cellulose tris(3,5-dichlorophenylcarbamate)-based chiral stationary phase, in a polar-organic mode of elution, enabled the successful enantioseparation of the investigated compounds. The developed chromatography method reveals a useful tool of monitoring in view of a proper forthcoming enantioselective synthetic protocol. Moreover, the optimized chromatographic conditions allowed the isolation of appropriate amounts of single enantiomers for the electronic circular dichroism studies that, coupled with in silico simulations, allowed assessing the absolute configuration of each species. [ABSTRACT FROM AUTHOR]

Among the mechanisms adopted by bacteria, efflux pumps (EPs) have been recognized as being significantly involved in contributing to resistance to commonly used antibacterial agents. However, little is known about their three-dimensional s tructures or the steric requirements for their inhibition. Lack of such knowledge includes NorA, one of the most studied Staphylococcus aureus EPs. In the present study, the use of two commercialized Cinchona alkaloid-based zwitterionic chiral stationary phases allowed the enantioseparation of four 2-((2-(4-propoxyphenyl)quinolin-4-yl)oxy)alkylamines 1 – 4 previously found to be potent S. aureus NorA efflux pump inhibitors when tested as racemates. In the identified optimal polar-ionic conditions (MeOH/THF/H 2 O-49/49/2 (v/v/v) + 25 mM formic acid, 12.5 mM diethylamine), repeated consecutive injections of 1 allowed the isolation of sufficient amount of its enantiomers (2.6 mg and 2.8 mg, for ( R )- 1 and ( S )- 1 , respectively) and then to evaluate their ability to inhibit the S. aureus NorA efflux pump. The biological evaluation highlighted the main contribution of the ( R )- 1 enantiomer to both the EtBr efflux inhibition and synergistic effect with against SA-1199B ( norA+ /A116E GrlA) respect to the racemate activity. The comparison between the experimental electronic circular dichroism and the time-dependent density functional theory calculations spectra of the two isolated enantiomeric fractions allowed for all compounds a clear and easy assignment of the enantiomeric elution order. [ABSTRACT FROM AUTHOR]

A chiral chromatography method enabling the simultaneous diastereo- and enantioseparation of N α -Boc- N 4 -(hydroorotyl)-4-aminophenylalanine [Boc-Aph(Hor)-OH, 1 ] was optimized with a quinine-based zwitterionic stationary phase. The polar-ionic eluent system consisting of ACN:MeOH:water—49.7:49.7:0.6 (v/v/v) with formic acid (4.0 mM) and diethylamine (2.5 mM), allowed the successful separation of the four acid stereoisomers: α d , d -/ d , l - 1 = 1.08; α d , l -/ l , d - 1 = 1.08; α l , d -/ l , l - 1 = 1.40. According to the in-house developed synthetic procedure and the recorded electronic circular dichroism spectra, the following stereoisomeric elution order was readily established in the optimal chromatographic conditions: d , d - 1 < d , l - 1 < l , d - 1 < l , l - 1 . With the aim of better understanding the molecular basis of the retention behaviour of the four stereoisomers in the employed chromatographic system and conditions, a computational protocol consisting in molecular dynamics simulations was applied. The use of the three descriptors INTER (in kcal mol −1 , encoding for the interaction energy between the selector SO unit and the whole system), INTER_SA (in kcal mol −1 , encoding for the interaction energy between SO and the sole selectand SA), and SELF (in kcal mol −1 , encoding for the conformational energy of SA relative to its minimum energy registered by the collected snapshots) revealed the active role of achiral sub-structural elements of the chiral stationary phase and eluent components in the overall stereorecognition mechanism. [ABSTRACT FROM AUTHOR]

A number of large-conductance calcium-activated potassium (BK) channel openers based on the 2-aryl-1,4-benzothiazine scaffold were previously synthesized, and 2-(5-bromo-2-methoxyphenyl)-6-trifluoromethyl-2 H -1,4-benzothiazin-3(4 H )-one ( 1 ) was identified as the most active compound. Since a stereoselective activation of BK channels was demonstrated for arylindolone derivatives, the effect of the absolute configuration at the C-2 position on the vasorelaxing potency of 2-aryl-1,4-benzothiazines is investigated in this article. Compound 1 was initially evaluated as a racemate: subsequently, the “racemic approach” was used to isolate its enantiomers. The excellent enantioresolution obtained using the Sepapak-4 column (CSP 4 , cellulose tris (4-chloro-3-metylphenylcarbamate); R S = 8.36; α = 2.03) allowed to collect highly pure enantiomeric fractions, with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively. Electronic circular dichroism (ECD) studies on the two isolated enantiomers, combined with time-dependent density functional theory (TD-DFT) calculations allowed to characterize the configuration of the enantiomers and determine a ( R ), ( S ) elution order. Results from biological assays indicated that the racemate and the isolated enantiomers are endowed with comparable vasorelaxing potency. [ABSTRACT FROM AUTHOR]

In the present study, the "Inverted Chirality Columns Approach (ICCA)" was applied to follow an asymmetric synthetic reaction, namely, the addition of butan-2-one to trans -β-nitrostyrene, catalysed by (S)-proline, leading to the formation of 3-methyl-4-phenyl-5-nitro-2-pentan-2-one. The ICCA method was applied to overcome the lack of pure enantiomeric standards. The two widely employed (R , R)- and (S , S)-Whelk-O1 chiral stationary phases (CSPs), incorporating fully synthesized enantiomeric chiral selectors, were profitably used for this purpose. The enantioselective analysis with the two CSPs was performed under optimized reversed-phase conditions with a water/acetonitrile (60/40, v/v) eluent. In the probe reaction under investigation, a diastereomeric excess >90% was found according to a well-established reaction mechanism, thus affording the enantiomer couple (3 S ,4 R)-3-methyl-4-phenyl-5-nitropentan-2-one and (3 R ,4 S)-3-methyl-4-phenyl-5-nitropentan-2-one as the main product. Therefore, the attention was exclusively focused on this enantiomers pair. Rather similar retention and separation factor [1.12 with (R , R)-Whelk-O1 and 1.13 with (S , S)-Whelk-O1] values as well as resolutions [2.06 with (R , R)-Whelk-O1 and 2.30 with (S , S)-Whelk-O1] were produced by the two enantiomeric CSPs. Applying the ICCA concept allowed to identify the two enantiomers-related peaks in the chromatograms, ultimately indicating a 65-to-35 enantiomeric per cent ratio. Electronic circular dichroism (ECD) and high-resolution mass spectrometry analyses of the two peaks collected during the enantioselective analyses further confirmed the enantiomeric nature of the identified compounds. The (3 S ,4 R) < (3 R ,4 S) enantiomer elution order with the (R , R)-Whelk-O1 was fully disclosed thanks to ECD studies coupled with in silico quantum mechanical simulations. As expected, reversed elution order turned out with (S , S)-Whelk-O1. Image 1 • The ICCA was applied to monitor an enantioselective synthesis without enantiomeric standards. • For the enantioseparation, the (R,R)- and (S,S)-Whelk-O1 CSPs were used under RP conditions. • Experimental and in silico ECD spectra defined the enantiomer elution order. • HRMS analysis facilitated the identification of the enantiomers on the chromatogram. [ABSTRACT FROM AUTHOR]

Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona -alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(−)] was successfully used to optimize the enantioresolution of compounds 1–3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)<(R) enantiomer elution order (EEO) was established for compounds 1 and 2 by analysing a pure enantiomeric standard of known configuration. This was not possible for 3 because not commercially available. For this compound, the same EEO was identified applying a procedure based on ab initio time-dependent density-functional theory simulations coupled to electronic circular dichroism analyses. Moreover, a molecular dynamics simulation unveiled the role of the phenolic OH in compound 3 in the retention mechanism. [Display omitted] • ZWIX(−) allows the enantioresolution of the three aromatic α-hydroxy acids. • The optimized polar-ionic conditions are fully compatible with MS detectors. • The developed LC method can be used to study biological matrices with MS detectors. • Combining in silico simulations and ECD analyses is useful to derive the EEO. • The retention mechanism can be explained with molecular dynamic simulations. [ABSTRACT FROM AUTHOR]

• (S,S)-Whek-O1 allowed the NP enantioseparation of partially saturated 2 H -indazoles. • (S,S)-Whek-O1 was used for a small-scale preparative enantioseparation. • (S,S)-Whek-O1 exhibited high enantioselectivity and chemoselectivity. • ECD studies coupled to ab initio TD-DFT simulations enabled to establish the EEO. • In-silico molecular dynamics can make a priori predictions on the enantioseparation. With the selection of partially saturated 2 H -indazoles as model compounds, we demonstrate the possibility to use Whelk-O1 chiral stationary phases (CSPs) to succeed in efficient small-scale preparative enantioseparations. Runs of three consecutive liquid chromatography injections (about 300 μg of racemate repeatedly injected in a 100 μL loop) produced groups of peaks without band contamination (α = 1.2 and R S = 2.57). With this procedure approximately 3.0 mg of each enantiomer, with enantiomeric excess ≥ 97% were obtained. Very profitably, the high volatility of n -hexane used as the sole eluent facilitated the solvent evaporation after the enantiomer recovery. High resolution mass spectrometry analysis confirmed that the chemical identity of the two enantiomers was preserved along the entire process. The ability of Whelk-O1 phases in enantioseparating structurally similar compounds was confirmed with the analysis of other two racemates. Moreover, the relevant chemoselectivity exhibited by the CSP towards the three racemates should allow to simultaneously optimizing the enantioselectivity of different analytes and perform small-scale enantioresolutions of different compounds during the same run. In this study , the integration of experimental off-line electronic circular dichroism analysis with ab initio time-dependent density-functional theory simulations facilitated the assignment of the absolute configuration of the single enantiomers, while a molecular dynamics protocol can be useful to make a priori predictions of the enantioseparation ability of CSP towards selected compounds. [ABSTRACT FROM AUTHOR]